Contrasting Enzymatic Activities of Topoisomerase IV and DNA Gyrase from Escherichia coli

Ullsperger, Chris; Cozzarelli, Nicholas R.

doi:10.1074/jbc.271.49.31549

Cited by 133 publications

(128 citation statements)

References 62 publications

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“…To further establish that the large products were catenated DNA rings, a preparation of catenated DNA was treated with Topo IV, which is a very active decatenating enzyme (21,22). In the presence of very low concentrations of Topo IV (125 pM), the large DNA products were partially resolved into intermediate bands and the monomer gDNA (Fig.…”

Section: Resultsmentioning

confidence: 99%

MukB-mediated Catenation of DNA Is ATP and MukEF Independent

Bahng

Hayama

Marians

2016

Journal of Biological Chemistry

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Edited by Patrick SungProperly condensed chromosomes are necessary for accurate segregation of the sisters after DNA replication. The Escherichia coli condesin is MukB, a structural maintenance of chromosomes (SMC)-like protein, which forms a complex with MukE and the kleisin MukF. MukB is known to be able to mediate knotting of a DNA ring, an intramolecular reaction. In our investigations of how MukB condenses DNA we discovered that it can also mediate catenation of two DNA rings, an intermolecular reaction. This activity of MukB requires DNA binding by the head domains of the protein but does not require either ATP or its partner proteins MukE or MukF. The ability of MukB to mediate DNA catenation underscores its potential for bringing distal regions of a chromosome together.The Escherichia coli chromosome is condensed about 1000-fold to fit into the nucleoid of the bacterium. A number of factors contribute to this extreme DNA condensation, among them are: DNA supercoiling, the binding of various nucleoid associated proteins like HU, Fis, and H-NS, and the binding of the structural maintenance of chromosomes (SMC) 3 -like condensin MukBEF (1, 2). SMC proteins act to manage the shape and behavior of chromosomes in both prokaryotes and eukaryotes (3). These proteins dimerize at a hinge region that is flanked by long coiled-coil regions that can be 40 -50 nm in length and that end in head domains that bind and hydrolyze ATP, as well as the bridging kleisin protein (MukF for the E. coli condensin (4)). The kleisin is then itself bound by another protein (MukE for the E. coli condensin (4)). There are three versions of the eukaryotic SMC proteins: the condensin, required for packaging of the chromosomes and comprised of SMC2 and SMC4; the cohesin, required for holding sister chromosomes together during mitosis and comprised of SMC1 and SMC3; and the SMC5-SMC6 complex, required for various aspects of DNA repair (3).It is generally acknowledged that the eukaryotic condensin and cohesin trap DNA topologically in the protein triangle formed by the SMC proteins and the kleisin (5, 6), although an alternative model for DNA binding for the eukaryotic cohesin exists (7). Recent reports suggest that the Bacillus subtilis SMC protein (8) and MukB (9) also trap chromosomes topologically. MukB binds linear and circular DNA in vitro and can induce negative supercoils and knots in relaxed circular DNA in the presence of a topoisomerase (10). Binding of MukB to chromosomal DNA in vivo requires ATP and MukEF (11, 12), although MukB DNA binding in vitro does not (10, 13).We (14) and the Burger and Oakley (15) labs have shown that the MukB hinge region interacts with the C-terminal ␤-propeller region of the ParC subunit of the cellular decatenase topoisomerase IV (Topo IV). We have reported (16) that this interaction stimulates the intramolecular activities of Topo IV, negative supercoil relaxation and knotting, but not the intermolecular activities of Topo IV, catenation/decatenation of DNA rings; whereas Berger, Oakley and colleag...

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Section: Resultsmentioning

confidence: 99%

MukB-mediated Catenation of DNA Is ATP and MukEF Independent

Bahng

Hayama

Marians

2016

Journal of Biological Chemistry

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“…The effect of DNA supercoiling on DNA unknotting by bacterial topoisomerase IV was investigated in 1996 by Ullsperger and Cozzarelli (27), who concluded that supercoiling of knots did not cause an appreciable increase in the rate of their unknotting by Topo IV. However, a closer look at their experimental data reveals that, although the unknotting rate of complex knots with nine crossings appears to be unaffected by supercoiling, there is a clear effect on unknotting of simple knots.…”

Section: Discussionmentioning

confidence: 99%

Tightening of DNA knots by supercoiling facilitates their unknotting by type II DNA topoisomerases

Witz

Dietler

Stasiak

2011

Proc. Natl. Acad. Sci. U.S.A.

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Using numerical simulations, we compare properties of knotted DNA molecules that are either torsionally relaxed or supercoiled. We observe that DNA supercoiling tightens knotted portions of DNA molecules and accentuates the difference in curvature between knotted and unknotted regions. The increased curvature of knotted regions is expected to make them preferential substrates of type IIA topoisomerases because various earlier experiments have concluded that type IIA DNA topoisomerases preferentially interact with highly curved DNA regions. The supercoiling-induced tightening of DNA knots observed here shows that torsional tension in DNA may serve to expose DNA knots to the unknotting action of type IIA topoisomerases, and thus explains how these topoisomerases could maintain a low knotting equilibrium in vivo, even for long DNA molecules.Brownian dynamics | DNA topology | DNA structure | DNA configurations D uring normal cell growth, various DNA transactions are facilitated by topoisomerase-mediated passage of one DNA segment through another. These strand passages can inadvertently lead to the formation of DNA knots (1-3), which are highly deleterious for living cells if not removed efficiently (4, 5). Type IIA DNA topoisomerases can use the free energy of ATP hydrolysis (6) to decrease the knotting level significantly below the equilibrium value for DNA rings subject to random passages between colliding segments (7). This topoisomerase (topo) IIA ability has been often assumed to be a physiologically relevant mechanism ensuring efficient DNA unknotting and decatenation (7-10), but doubts on this relevance have been raised as well (11). Particularly problematic is the fact that the original study establishing that type IIA DNA topoisomerases actively unknot DNA also revealed that this ability decreases sharply with increasing DNA length. Although based on only two plasmid sizes, that study showed the generality of this length dependence for several topoisomerases (7) and, therefore, active unknotting would be doomed to be ineffective for long DNA molecules, where random passages lead to very frequent knotting (12). While trying to understand why earlier experimental studies predicted ineffectiveness of type IIA DNA topoisomerases in unknotting of long DNA molecules, we recognized that these studies were performed using torsionally relaxed DNA (7), whereas naturally occurring DNA is frequently under torsional tension (13), the best-known examples of this state being negatively supercoiled bacterial plasmids. To investigate the potential effects of torsional constraints on the activity of type IIA topoisomerases, we have performed Brownian dynamics simulations to examine how negative supercoiling affects the structure of knotted DNA molecules and whether DNA supercoiling can cause knotted regions to be recognized specifically and then unknotted by topoisomerases. We observed that DNA supercoiling results in the tightening of knots, which leads directly to an increase in curvature of the knotted regions, in turn re...

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“…Recent studies further sustain its role as a decatenase by demonstrating that, through the interaction with a septal ring protein FtsK, Topo IV is only accessible to DNA at the end of replication (14). Because the decatenating activity of Topo IV is stimulated by (Ϫ) superhelical density, the two enzymes appear to work in concert to complete replication (15).…”

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Structure of the Topoisomerase IV C-terminal Domain

Hsieh

Farh

Huang

et al. 2004

Journal of Biological Chemistry

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Bacteria possess two closely related yet functionally distinct essential type IIA topoisomerases (Topos). DNA gyrase supports replication and transcription with its unique supercoiling activity, whereas Topo IV preferentially relaxes (؉) supercoils and is a decatenating enzyme required for chromosome segregation. Here we report the crystal structure of the C-terminal domain of Topo IV ParC subunit (ParC-CTD) from Bacillus stearothermophilus and provide a structure-based explanation for how Topo IV and DNA gyrase execute distinct activities. Although the topological connectivity of ParC-CTD is similar to the recently determined CTD structure of DNA gyrase GyrA subunit (GyrA-CTD), ParC-CTD surprisingly folds as a previously unseen broken form of a six-bladed ␤-propeller. Propeller breakage is due to the absence of a DNA gyrase-specific GyrA box motif, resulting in the reduction of curvature of the proposed DNA binding region, which explains why ParC-CTD is less efficient than GyrA-CTD in mediating DNA bending, a difference that leads to divergent activities of the two homologous enzymes. Moreover, we found that the topology of the propeller blades observed in ParC-CTD and GyrA-CTD can be achieved from a concerted ␤-hairpin invasion-induced fold change event of a canonical six-bladed ␤-propeller; hence, we proposed to name this new fold as "hairpininvaded ␤-propeller" to highlight the high degree of similarity and a potential evolutionary linkage between them. The possible role of ParC-CTD as a geometry facilitator during various catalytic events and the evolutionary relationships between prokaryotic type IIA Topos have also been discussed according to these new structural insights.

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Contrasting Enzymatic Activities of Topoisomerase IV and DNA Gyrase from Escherichia coli

Cited by 133 publications

References 62 publications

MukB-mediated Catenation of DNA Is ATP and MukEF Independent

MukB-mediated Catenation of DNA Is ATP and MukEF Independent

Tightening of DNA knots by supercoiling facilitates their unknotting by type II DNA topoisomerases

Structure of the Topoisomerase IV C-terminal Domain

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